The overall goal of the project is to define the regulatory mechanisms of synthesis, assembly and membrane insertion of small intestinal oligosaccharidases that are crucial for the assimilation of dietary carbohydrate. Sucrase-Isomaltase (S-I), a hybrid enzyme that is essential for intestinal surface hydrolysis of sucrose and alpha-limit dextrin products from starch, will be isolated from rat intestinal brush border, and a monospecific antibody, raised in rabbits, will be used to establish a solid-phase radioimmunoassay. The subunit structure of S-I will be determined under varying denaturing conditions and the composition of the brush border enzyme compared with that of the cytosol which is known to contain incomplete active sucrase protein that may constitute a precursor of the brush border enzyme. The sequence of synthesis and assembly of sucrase-isomaltase in the cytosol compartment and on intracellular membranes and organelles (ribosomes, endoplasmic reticulum, Golgi and laterobasal membrane) will be examined after pulse labeling with (3H)leucine or (3H)fucose and isolation of immunoreactive sucrase protein. The role of the carbohydrate moiety of sucrase-isomaltase in this synthesis-assembly process will be examined by inhibiting glycosylation in vivo with Tunicamycin prior to pulse labeling. Finally, human intestinal lactase will be purified and a monospecific antiserum obtained from rabbits. Solubilized intestinal tissues from lactase-deficient individuals will be examined by immunodiffusion, immunoelectrophoresis and solid-phase specific radioimmunoassay to identify a putative enzymatically inactive variant of the lactase protein. If this is identified, comparative structural studies of the normal and aberrant enzyme will be carried out.